Conformation of acetylcholine receptor in the presence of agonists and antagonists

The conformations of acetylcholine receptor fromTorpedo californica in the absence and presence of agonists, antagonists, and local anesthetics were studied by circular dichroism (CD). Without ligands, the receptor had about 40% helix, 20% -sheets, and 10% -turns as analyzed from its far-UV CD spectrum. Its near-UV CD spectrum resembled that of acetylcholinesterase from the same source. None of the ligands studied altered the far-UV spectrum of the receptor. However, in the near-UV region, carbamylcholine and acetylcholine shifted the Phe and Tyr bands of AChR to less negative, whereas hexamethonium changed the Tyr bands to more negative, indicating that the site of binding of agonists and antagonists and their effect on the conformation of the receptor may be different. Decamethonium, procaine, and lidocaine had no effect on both the far- and near-UV CD spectra of acetylcholine receptor.     

Modulation of Calcium Fluxes Across Synaptosomal Plasma Membrane by Local Anesthetics

We have studied the effects of local anesthetics (dibucaine, tetracaine, lidocaine, and procaine) on calcium fluxes through the plasma membrane of synaptosomes. All these local anesthetics inhibit the ATP-dependent calcium uptake by inverted plasma membrane vesicles at concentrations close to those that promote an effective blockade of the action potential. The values obtained for the K0.5 of inhibition of calcium uptake are the following: 23 microM (dibucaine), 0.44 mM (lidocaine), 1.5 mM (procaine), and 0.8 mM (tetracaine). There is a good correlation between these K0.5 values and the concentrations of the local anesthetics that inhibit the Ca2(+)-dependent Mg2(+)-ATPase of these membranes. In addition, except for procaine, these local anesthetics stimulate severalfold the Ca2+ outflow via the Na+/Ca2+ exchange in these membranes. This effect, however, is observed at concentrations slightly higher than those that effectively inhibit the ATP-dependent Ca2+ uptake, e.g., 80-700 microM dibucaine, 2-10 mM lidocaine, and 1-3 mM tetracaine. The results suggest that the Ca2+ buffering of neuronal cytosol is altered by these anesthetics at pharmacological concentrations.     

Procaine rapidly inactivates acetylcholine receptors from Torpedo and competes with agonist for inhibition sites

The relationship between the high-affinity procaine channel inhibition site (apparent dissociation constant Kp congruent to 200 microM) and the agonist self-inhibition site on acetylcholine receptors (AChRs) from Torpedo electroplaque was investigated by using rapid 86Rb+ quenched-flux assays at 4 degrees C in native AChR-rich vesicles on which 50-60% of ACh activation sites were blocked with alpha-bungarotoxin (alpha-BTX). In the presence of channel-activating acetylcholine (ACh) concentrations (10 microM-10 mM) alone, AChR undergoes one phase of inactivation (fast desensitization, rate = kd) in under a second. Addition of procaine produces two-phase inactivation similar to that seen with self-inhibiting (greater than 10 mM) ACh concentrations [Forman & Miller (1988) Biophys. J. 54, 149-158]--rapid inactivation (rate = kr) complete in 30-75 ms is followed by fast desensitization at the same kd observed without procaine. The dependence of kr on [procaine] is consistent with a bimolecular association between procaine and its AChR site with kon = 2.5 X 10(5) M-1 s-1, koff = 36 s-1, and Kp = 145 +/- 36 microM). Inhibition of AChR function by mixtures of procaine (up to 12Kp) plus self-inhibiting concentrations of ACh or suberyldicholine ([SubCh] up to 13 X the 50% self-inhibiting agonist concentration, KB) was studied by reducing the level of alpha-BTX block in vesicles. The apparent KB increased in the presence of procaine, and the apparent KP increased linearly with [SubCh], indicating mutually exclusive actions at a common AChR site. Our data support a mechanism where procaine binds preferentially to the open-channel AChR state, since no procaine-induced inactivation is observed without agonist and kr's dependence on [ACh] in the channel-activating range closely parallels that of 86Rb+ flux response to ACh.     

Reaction of [3H]meproadifen mustard with membrane-bound Torpedo acetylcholine receptor

The Torpedo nicotinic acetylcholine receptor (AChR) contains a binding site for aromatic amine noncompetitive antagonists that is distinct from the binding site for agonists and competitive antagonists. To characterize the location and function of this allosteric antagonist site, an alkylating analog of meproadifen has been synthesized, 2-(chloroethylmethylamino)-ethyl-2, 2-diphenylpentanoate HCl (meproadifen mustard). Reaction of [3H]meproadifen mustard with AChR-rich membrane suspensions resulted in specific incorporation of label predominantly into the AChR alpha-subunit with minor incorporation into the beta-subunit. Specific labeling required the presence of high concentration of agonist and was inhibited by reversible noncompetitive antagonists including proadifen, meproadifen, perhydrohistrionicotoxin (HTX), and tetracaine when present at concentrations consistent with the binding affinity of these compounds for the allosteric antagonist site. No specific alkylation of the AChR alpha-subunit was detected in the absence of agonist, or in the presence of the partial agonist phenyltrimethylammonium or the competitive antagonists, d-tubocurarine, gallamine triethiodide, or decamethonium. Reaction with 35 microM meproadifen mustard for 70 min in the presence of carbamylcholine produced no alteration in the concentration of [3H]ACh-binding sites, but decreased by 38 +/- 4% the number of allosteric antagonist sites as measured by [3H]HTX binding. This decrease was not observed when the alkylation reaction was blocked by the presence of HTX. These results lead us to conclude that meproadifen mustard alkylates the allosteric antagonist site in the Torpedo AChR and that part of that site is associated with the AChR alpha-subunit.     

High affinity binding of alpha-bungarotoxin to the purified alpha-subunit and to its 27,000-dalton proteolytic peptide from Torpedo marmorata acetylcholine receptor. Requirement for sodium dodecyl sulfate

Intact nicotinic acetylcholine receptor (AChR) tightly binds alpha-bungarotoxin. The two toxin-binding sites are presumed to be on the two alpha-subunits, either on or near the ACh-binding sites. Isolated alpha-subunits have been found to maintain weak binding to alpha-bungarotoxin (KD approximately 0.2 microM). We describe here conditions under which the alpha-subunit and a 27,000-dalton proteolytic peptide bound alpha-bungarotoxin with high affinity. The four subunits of Torpedo marmorata AChR, as well as several proteolytic peptides of the alpha-subunit, were first purified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. We found that the purified alpha-subunit (but not the beta-, gamma- or delta-subunits) and its 27,000-dalton peptide specifically bound 125I-labeled alpha-bungarotoxin with KD approximately 3 and 6 nM, i.e., about two orders of magnitude lower than the intact AChR. Nearly 100% of the sites were recovered. The recovery of this high affinity binding required the presence of SDS (approximately 0.02%) but non-denaturing detergents had a strongly inhibitory effect. Unlabeled alpha-toxins competed with labeled alpha-bungarotoxin, alpha-bungarotoxin being more effective than all the other toxins tested. Decamethonium and hexamethonium competed efficiently with alpha-bungarotoxin binding but carbamylcholine had only a weak effect. The main immunogenic region of the AChR was only partially preserved since conformation-dependent monoclonal antibodies to this region bound the alpha subunit-toxin complexes, but much less efficiently than the intact AChR. We conclude that SDS can be advantageous to the recovery of high toxin binding to the alpha subunit which still has not completely recovered its native conformation.     

Detection of nicotinic receptor ligands with a light addressable potentiometric sensor.

The nicotinic acetylcholine receptor, purified from Torpedo electric organ, was coupled to a light addressable potentiometric sensor (LAPS) to form a LAPS-receptor biosensor. Receptor-ligand complexes containing biotin and urease were captured on a biotinylated nitrocellulose membrane via a streptavidin bridge and detected with a silicon-based sensor. Competition between biotinylated alpha-bungarotoxin and nonbiotinylated ligands formed the basis of this assay. This biosensor detected both agonists (acetylcholine, carbamylcholine, succinylcholine, suberyldicholine, and nicotine) and competitive antagonists (d-tubocurarine, alpha-bungarotoxin, and alpha-Naja toxin) of the receptor with affinities comparable to those obtained using radioactive ligand binding assays. Consistent with agonist-induced desensitization of the receptor, the LAPS-receptor biosensor reported a time-dependent increase in affinity for the agonist carbamylcholine as expected, but not for the antagonists.     

Influence of agonists and antagonists on the segmental motion of residues near the agonist binding pocket of the acetylcholine-binding protein

Using the Lymnaea acetylcholine-binding protein as a surrogate of the extracellular domain of the nicotinic receptor, we combined site-directed labeling with fluorescence spectroscopy to assess possible linkages between ligand binding and conformational dynamics. Specifically, 2-[(5-fluoresceinyl)aminocarbonyl]ethyl methanethiosulfonate was conjugated to a free cysteine on loop C and to five substituted cysteines at strategic locations in the subunit sequence, and the backbone flexibility around each site of conjugation was measured with time-resolved fluorescence anisotropy. The sites examined were in loop C (Cys-188 using a C187S mutant), in the beta9 strand (T177C), in the beta10 strand (D194C), in the beta8-beta9 loop (N158C and Y164C), and in the beta7 strand (K139C). Conjugated fluorophores at these locations show distinctive anisotropy decay patterns indicating different degrees of segmental fluctuations near the agonist binding pocket. Ligand occupation and decay of anisotropy were assessed for one agonist (epibatidine) and two antagonists (alpha-bungarotoxin and d-tubocurarine). The Y164C and Cys-188 conjugates were also investigated with additional agonists (nicotine and carbamylcholine), partial agonists (lobeline and 4-hydroxy,2-methoxy-benzylidene anabaseine), and an antagonist (methyllycaconitine). With the exception of the T177C conjugate, both agonists and antagonists perturbed the backbone flexibility of each site; however, agonist-selective changes were only observed at Y164C in loop F where the agonists and partial agonists increased the range and/or rate of the fast anisotropy decay processes. The results reveal that agonists and antagonists produced distinctive changes in the flexibility of a portion of loop F.     

Is agonist self-inhibition at the nicotinic acetylcholine receptor a nonspecific action?

Agonist concentration-response relationships at nicotinic postsynaptic receptors were established by measuring 86Rb+ efflux from acetylcholine receptor rich native Torpedo membrane vesicles under three different conditions: integrated net ion efflux (in 10 s) from untreated vesicles, integrated net efflux from vesicles in which most acetylcholine sites were irreversibly blocked with alpha-bungarotoxin, and initial rates of efflux (5-100 ms) from vesicles that were partially blocked with alpha-bungarotoxin. Exposure to acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, or (-)-nicotine over 10(8)-fold concentration ranges results in bell-shaped ion flux response curves due to stimulation of acetylcholine receptor channel opening at low concentrations and inhibition of channel function at 60-2000 times higher concentrations. Concentrations of agonists that inhibit their own maximum 86Rb+ efflux by 50% (KB values) are 110, 211, 3.0, 39, and 8.9 mM, respectively, for the agonists listed above. For acetylcholine and carbamylcholine, KB values determined from both 10-s and 15-ms efflux measurements are the same, indicating that the rate of agonist-induced desensitization increases to maximum at concentrations lower than those causing self-inhibition. For all partial and full agonists studied, Hill coefficients for self-inhibition are close to 1.0. Concentrations of agonists up to 8 times KB did not change the order parameter reported by a spin-labeled fatty acid incorporated in Torpedo membranes. We conclude that agonist self-inhibition cannot be attributed to a general nonspecific membrane perturbation. Instead, these results are consistent with a saturable site of action either at the lipid-protein interface or on the acetylcholine receptor protein itself.     

The hydrophobic photoreagent 3-(trifluoromethyl)-3-m-([125I] iodophenyl) diazirine is a novel noncompetitive antagonist of the nicotinic acetylcholine receptor

We have shown previously that the lipophilic photoreagent 3-(trifluoromethyl)3-m-([125I]iodophenyl)-diazirine ([125I]TID) photolabels all four subunits of the Torpedo nicotinic acetylcholine receptor (AChR) and that greater than 70% of this photoincorporation is inhibited by cholinergic agonists and some noncompetitive antagonists, including histrionicotoxin (HTX), but not phencyclidine (PCP; White, B.H., and Cohen, J.B. (1988) Biochemistry 27, 8741-8751). We have now examined the effects of nonradioactive TID on (a) AChR photoincorporation of [125I]TID, (b) AChR-mediated ion transport, and (c) AChR binding of several cholinergic ligands. We find that TID inhibits [125I]TID photoincorporation into the AChR to the same extent as carbamylcholine. The saturable component of [125I]TID photolabeling is half-maximal at 4 microM [125I]TID with 0.5 mol specifically incorporated per mol of AChR after 30 min photolysis with 60 microM [125I]TID. Repeated labeling of membranes at a fixed [125I]TID concentration gave results consistent with a maximal incorporation of one [125I]TID molecule per AChR. Nonradioactive TID also noncompetitively inhibits agonist-stimulated 22Na+ efflux from Torpedo vesicles with an IC50 of 1 microM. Furthermore, TID inhibits allosterically the binding of [3H]HTX, decreasing its affinity for the AChR 5-fold both in the presence and absence of agonist. In contrast, TID has little effect on [3H]PCP binding in the absence of agonist but completely inhibits it in the presence of agonist. TID enhances the cooperativity of [3H]nicotine binding. [125I]TID is thus a photoaffinity label for a novel noncompetitive antagonist binding site on the AChR that is linked allosterically to the binding sites of both agonists and other noncompetitive antagonists. The [125I]TID site is presumably located within the central pore of the AChR.     

Fine localization of the major alpha-bungarotoxin binding site to residues alpha 189-195 of the Torpedo acetylcholine receptor. Residues 189, 190, and 195 are indispensable for binding

alpha-Bungarotoxin blocks acetylcholine-mediated ion channel opening of peripheral acetylcholine receptors (AChR). A major binding region for alpha-bungarotoxin has been recently identified within parts of the segment 170-204 of the alpha-subunit. We used the Pepscan systematic peptide synthesis system to determine the minimum Torpedo AChR segment required for alpha-bungarotoxin binding and to investigate the role of each residue within this segment. Continuously overlapping decapeptides within alpha 179-203 and several decapeptides covering other alpha-subunit sequences showed that alpha 188-197 and alpha 189-198 exhibited the best 125I-alpha-bungarotoxin binding activity (KD = 7.3 x 10(-8) and 4.3 x 10(-8) M, respectively). Several continuously overlapping nona-, octa-, hepta-, hexa-, and tetrapeptides showed that the heptapeptide alpha 189-195 was the minimum sequence with high binding activity (KD = 5.6 x 10(-8)M). d-Tubocurarine, but not carbamylcholine, blocked toxin binding. Twenty-six analogs of the alpha 188-197, most having 1 residue substituted by Ala or Gly, showed that Tyr189, Tyr190, and especially Asp195 were indispensable for 125I-alpha-bungarotoxin binding. Cys192 and Cys193 could be substituted by other amino acids, proving that the disulfide bond between alpha 192-193 was not required for alpha-bungarotoxin binding. The decreased alpha-bungarotoxin binding capacity of the equivalent human muscle AChR alpha 188-197 peptide was the result of substitution of Tyr by Thr at alpha 189.     

Effects of lipids and detergents on the conformation of the nicotinic acetylcholine receptor from Torpedo californica.

The hydrophobic, photoreactive probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) was used to characterize the effects of lipids and detergents on acetylcholine receptor (AChR) conformation. Affinity purified AChR reconstituted into dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidic acid (DOPA), and cholesterol showed the same pattern of [125I]TID-labeling and demonstrated the same reduction in labeling of all four subunits upon desensitization by the agonist carbamylcholine, as partially purified AChR in native lipids. On the basis of the patterns of [125I]TID incorporation, reconstitution into DOPC/DOPA also appeared to stabilize the resting (functional) conformation of the AChR, while reconstitution in DOPC/cholesterol or DOPC alone largely desensitized the AChR. The effects of lipids on the functional state of the AChR was determined independently by measuring the ability of AChR reconstituted into different lipid combinations to undergo the change in affinity for agonist diagnostic of desensitization. The dramatic reduction in the apparent levels of [125I]TID associated with the subunits of the AChR observed upon agonist-induced desensitization was shown not to be due to a change in affinity for tightly bound lipid. Solubilization of affinity purified AChR reconstituted into DOPC/DOPA/cholesterol by the non-ionic detergents octyl glucoside, Triton X-100, and Tween 20 (final detergent concentration = 1%) was shown to produce the same pattern of [125I]TID-labeling as desensitization by agonist, while solubilization in 1% sodium cholate appeared to stabilize a conformation of the AChR more similar to the resting state.     

Characterization of an adult muscle acetylcholine receptor subunit by expression in fibroblasts.

To study the functional and structural roles of the epsilon subunit in adult muscle acetylcholine receptor (AChR), we have co-expressed the alpha and epsilon subunits of the mouse receptor in transfected fibroblasts. Ligand binding studies suggest that association of epsilon with alpha subunit results in a lower association rate constant for 125I-labeled alpha-bungarotoxin binding than that of the unassembled alpha subunit, approaching that for toxin binding to the AChR. Furthermore, alpha epsilon complexes contain high affinity binding sites for competitive antagonists and agonists not present in the unassembled alpha subunit, but similar to one of the two nonequivalent binding sites in the adult AChR. Structural analysis of alpha epsilon complexes by sucrose gradient velocity centrifugation suggests that some of the complexes formed are trimers or tetramers of alpha and epsilon subunits. Comparison of these data with those previously obtained for alpha gamma complexes suggests that gamma and epsilon have homologous functional roles and identical structural positions in the fetal and adult AChRs, respectively.     

Characterization of the channel properties of a neuronal acetylcholine receptor reconstituted into planar lipid bilayers

An alpha-toxin-binding membrane protein, isolated from the head and thoracic ganglia of the locus (Locusta migratoria), was reconstituted into planar lipid bilayers. Cholinergic agonists such as acetylcholine, carbamylcholine, and suberyldicholine induced fluctuations of single channels, which suggests that the protein represents a functional cholinergic receptor channel. The antagonist d-tubocurarine blocked the activation of the channels, whereas hexamethonium had only a weak effect; similar properties have been described for nicotinic insect receptors in situ. The channel was selectively permeable to monovalent cations but was impermeable to anions. The conductance of the channel (75 pS in 100 mM NaCl) was independent of the type of agonist used to activate the receptor. Kinetic analysis of the channel gating revealed that, at high agonist concentrations (50 microM carbamylcholine), more than one closed state exists and that multiple gating events, bursting as well as fast flickering, appeared. At very high agonist concentrations (500 microM carbamylcholine), desensitization was observed. Channel kinetics were dependent on the transmembrane potential. Comparing the conductance, the kinetics, and the pharmacology of nicotinic acetylcholine receptor from insect ganglia and fish electroplax reconstituted into bilayers revealed obvious similarities but also significant differences.     

Agonists block currents through acetylcholine receptor channels

We have examined the effects of high concentrations of cholinergic agonists on currents through single acetylcholine receptor (AChR) channels on clonal BC3H1 cells. We find that raised concentrations of acetylcholine (ACh; above 300 microM) or carbamylcholine (Carb; above 1,000 microM) produce a voltage- and concentration-dependent reduction in the mean single-channel current. Raised concentrations of suberyldicholine (Sub; above 3 microM) produce a voltage- and concentration-dependent increase in the number of brief duration low-conductance interruptions of open-channel currents. These observations can be quantitatively described by a model in which agonist molecules enter and transiently occlude the ion-channel of the AChR.     

Interaction of local anesthetics with Torpedo californica membrane-bound acetylcholine receptor

The effects of local anesthetics on the rate of the agonist-induced increase in ligand affinity of membrane-bound acetylcholine receptor from Torpedo californica were examined. The rate of the transition in receptor affinity was determined by following the time-dependent increase in inhibition of iodinated alpha-bungarotoxin binding caused by 1 microM carbamylcholine. At concentrations below those that directly inhibited the binding of iodinated alpha-bungarotoxin, dibucaine increased the rate of the transition to a high-affinity state and tetracaine decreased this rate. The measured rate constants were 0.026 +/- 0.008 s-1 in the presence and 0.010 +/- 0.002 s-1 in the absence of dibucaine while tetracaine decreased the rate to 0.006 +/- 0.002 s-1 as compared to a control value of 0.012 +/- 0.003 s-1. A parallel was observed between the effectiveness of a compound in increasing or decreasing the rate of the agonist-induced transition in affinity and the change in its apparent inhibition constant in the presence of carbamylcholine (increase or decrease) measured by the displacement of tritiated perhydrohistrionicotoxin. This parallel could be explained by assuming (a) that local anesthetics bound directly to the specific histrionicotoxin binding site or (b) that they bound to a different site and the observed effects were caused by conformational changes.     

Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor

We have stably expressed in fibroblasts different pairs of alpha and non-alpha subunits of the mouse muscle nicotinic acetylcholine receptor (AChR). The gamma and delta, but not the beta, subunits associated efficiently with the alpha subunit, and they extensively modified its binding characteristics. The alpha gamma and alpha delta complexes formed distinctly different high affinity binding sites for the competitive antagonist d-tubocurarine that, together, completely accounted for the two nonequivalent antagonist binding sites in native AChR. The alpha delta complex and native AChR had similar affinities for the agonist carbamylcholine. In contrast, although the alpha gamma complex contains the higher affinity competitive antagonist binding site, it had an affinity for carbamylcholine that was an order of magnitude less than that of the alpha delta complex or the AChR. The comparatively low agonist affinity of the alpha gamma complex may represent an allosterically regulated binding site in the native AChR. These data support a model of two nonequivalent binding sites within the AChR and imply that the basis for this nonequivalence is the association of the alpha subunit with the gamma or delta subunit.     

Measuring relative acetylcholine receptor agonist binding by selective proton nuclear magnetic resonance relaxation experiments.

A method is presented that uses selective proton Nuclear Magnetic Resonance (NMR) relaxation measurements of nicotine in the presence of the acetylcholine receptor to obtain relative binding constants for acetylcholine, carbamylcholine, and muscarine. For receptors from Torpedo californica the results show that (a) the binding constants are in the order acetylcholine greater than nicotine greater than carbamylcholine greater than muscarine; (b) selective NMR measurements provide a rapid and direct method for monitoring both the specific and nonspecific binding of agonists to these receptors and to the lipid; (c) alpha-bungarotoxin can be used to distinguish between specific and nonspecific binding to the receptor; (d) the receptor--substrate interaction causes a large change in the selective relaxation time of the agonists even at concentrations 100x greater than that of the receptor. This last observation means that these measurements provide a rapid method to monitor drug binding when only small amounts of receptor are available. Furthermore, the binding strategies presented here may be useful for the NMR determination of the conformation of the ligand in its bound state.     

Induction of phosphorylation and cell surface redistribution of acetylcholine receptors by phorbol ester and carbamylcholine in cultured chick muscle cells

We have investigated the mechanisms regulating the clustering of nicotinic acetylcholine receptor (AChR) on the surface of cultured embryonic chick muscle cells. Treatment of these cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, was found to cause a rapid dispersal of AChR clusters, as monitored by fluorescence microscopy of cells labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin. The loss of AChR clusters was not accompanied by an appreciable change in the amount of AChR on the surface of these cells, as measured by the specific binding of [125I]Bgt. Analysis of the phosphorylation pattern of immunoprecipitable AChR subunits showed that the gamma- and delta-subunits are phosphorylated by endogenous protein kinase activity in the intact muscle cells, and that the delta-subunit displays increased phosphorylation in response to TPA. Structural analogues of TPA which do not stimulate protein kinase C have no effect on AChR surface topography or phosphorylation. Exposure of chick myotubes to the cholinergic agonist carbamylcholine was found to cause a dispersal of AChR clusters with a time course similar to that of TPA. Like TPA, carbamylcholine enhances the phosphorylation of the delta-subunit of AChR. The carbamylcholine-induced redistribution and phosphorylation of AChR is blocked by the nicotinic AChR antagonist d-tubocurarine. TPA and carbamylcholine have no effect on cell morphology during the time-course of these experiments. These findings indicate that cell surface topography of AChR may be regulated by phosphorylation of its subunits and suggest a mechanism for dispersal of AChR clusters by agonist activation.     

A study of the mechanism by which some amphiphilic drugs affect human erythrocyte acetylcholinesterase activity.

The effects of the local anaesthetics procaine, tetracaine and lidocaine and of the antidepressant imipramine on human erythrocyte acetylcholinesterase were investigated. All four amphiphilic drugs inhibited enzymic activity, the IC50 (the concentration causing 50% inhibition) being about 0.40 mM for procaine, 0.05 mM for tetracaine, 0.70 mM for imipramine and 7.0 mM for lidocaine. Procaine and tetracaine inhibited acetylcholinesterase activity competitively at concentrations at which they did not perturb the physical state of the membrane lipid environment, as assessed by steady-state fluorescence polarization, whereas lidocaine and imipramine displayed mixed inhibition kinetics at concentrations at which they induced an enhancement of membrane fluidity. The question was addressed as to whether membrane integrity is a prerequisite for imipramine and lidocaine action. Membrane solubilization by 1% Triton X-100 and a decrease, by dilution, in the detergent concentration to 0.05% [which is above the Triton X-100 critical micelle concentration (c.m.c.)] did not substantially affect the inhibitory potency of the two amphiphilic drugs at their IC50; in the presence of increasing detergent concentrations the inhibitory potency of imipramine was gradually decreased, but not abolished, whereas the inhibitory effect of lidocaine was only slightly diminished, even at 1% Triton X-100. It is suggested that neither competitive nor mixed inhibition kinetics arise from conformational changes of the protein driven by a modification of the physical state of the lipid environment, but from a direct interaction of the amphiphilic drugs with acetylcholinesterase. In particular, the partial loss of the inhibitory potency of imipramine and lidocaine that is observed upon increasing Triton X-100 concentration well above its c.m.c. could be explained in terms of amphiphile partition in detergent micelles and, in turn, of a decreased effective concentration of the two inhibitors in the aqueous phase.